Established anticancer chemotherapeutics (such as cisplatin, doxorubicin, fluorouracil) are injected into the bloodstream or administered as tablets by swallowing. To successfully treat cancer cells the patient must receive high doses of chemotherapeutics. Because the conventional chemotherapeutics are not able to distinguish well between cancerous or healthy tissue, the patient usually suffers from strong side effects.
Recent strategies in chemotherapy are based on the principle of transporting the chemotherapeutics directly to the cancerous cells and therefore specifically attack the cancer cells while the healthy cells survive. Cancerous cells exhibit modified proteins on the cell membrane that enables a targeted recognition of the cells. With so-called antibody-drug conjugates (Chari et al., 2014), one active chemotherapeutic molecule per antibody can be transported to cancer cells. To enhance the efficiency of the targeted transport, nanoparticles have attracted increasing attention. For example, mesoporous silica particles, polymer constructs, liposomes, dendrimers or DNA origami objects have been investigated in this context. Nanoparticles can be transported to cancer cells either passively (e.g. through the EPR-effect) or they can interact more specifically with certain cell types through targeting. Nanoparticles can be taken up by cancer cells through endocytosis. The internalized particles or their cargo must then escape from the endosome to release the encapsulated drug and to kill the cancer cell. Generally, in case of transport through nanoparticles, the delivery system remains in the cell and must be degraded or removed from the body as extrinsic material. Furthermore, the toxic substances delivered to the cancer cells remain in the body and could lead to damage before their removal or if they leak out of the drug delivery system. In addition to epithelial cancer cells, mesenchymal cancer cells present a serious issue in oncology. They enable the tumor to recover after conventional chemotherapy and may spread the tumor all over the body by creating metastases. Only very few substances are known for the successful treatment of this mesenchymal cancer cell type (e. g. salinomycin, etoposide, abamectin, nigericin) (Gupta et al., 2009).
The transport system, so called drug delivery vehicles, benefits from certain characteristics such as high porosity/capacity for efficient loading with drug molecules, good biocompatibility and biodegradability, and a suitable size and shape for efficient cell uptake. Several materials, such as mesoporous silica particles, liposomes, polymer constructs, or dendrimers have been published that are intended to combine the above requirements within one drug delivery system.
These systems need to be designed such that premature release of the toxic drugs is prevented. As mentioned above, targeting, endocytosis and endosomal escape need to be achieved to ensure effective delivery of the drugs to the target cells. Specifically, endosomal escape has been achieved with photochemical methods (Mackowiak et al., 2013; Schlossbauer et al., 2012), temperature dependent mechanisms (Schlossbauer et al., 2010) or pH-responsive systems (Varkouhi et al., 2011; Behr et al., 1997).
Generally, the backbone of the drug delivery system remains in the body and must be degraded and removed from the body as extrinsic material.
Therefore, a major goal in the development of drug delivery systems is to increase the biocompatibility of the backbone. As an example, the PEGylation of silica nanoparticles has been investigated to eliminate hemolysis (Lin and Haynes 2010) or to avoid the activation of the immune system (He et al., 2010).
The utilization of drug delivery systems opens up many possibilities in cancer treatment, for example: the protection and transport of sensible drug molecules, the easy exchangeability of drugs, the reduction of dosage, the increase of circulation time, the increase of drug concentration at the targeted site, and the opportunity to design a personalized medication. Generally speaking, it would constitute a major advance in drug delivery if highly toxic substances could be avoided altogether.
Recently, apatite Ca10(PO4)6(OH)2, the main inorganic component of natural bone and teeth, has been discussed as a new promising platform for advanced drug delivery applications due to its high biocompatibility and non-toxicity (Iafisco et al., 2009; Dorozhkin and Epple, 2002; Palmer et al., 2008). Furthermore, apatite is known to be biodegradable (Arcos and Vallet-Regi, 2013) and as a result could solve the problem of eliminating the transport material from the body. However, calcium phosphate-based materials suffer from some drawbacks regarding applications in drug delivery systems. The maximum known surface area for calcium phosphate-based compounds is published to be 315 m2 g−1 which limits the loading capacity (Chen et al., 2014). Nevertheless, porous calcium phosphate based compounds were loaded with docetaxel (Chen et al., 2014; Ding et al., 2015), silybin (Chen et al., 2015) ibuprofen (Zhao et al., 2012) and doxorubicin (Rodriguez-Ruiz et al., 2013).
Next to loading drugs into the network of porous calcium phosphate particles, there have been investigations on co-precipitation methods of Ca2+- and PO43−-ions with Gemcitabine, siRNA, or proteins (Li et al., 2012; Zhang et al., 2013). The co-precipitated particles were delivered to cells, endosomal release due to the proton sponge effect was observed, and the function of the drug was proven. With an approach like this, the used molecules must be stable and soluble under the reaction conditions of co-precipitation. Because most drug molecules are not very soluble in water and therefore not suitable for water based co-precipitation methods, the authors (Li et al., 2012; Zhang et al., 2013) introduced a water-in-oil based synthesis approach.
However, if an exchange of the drug is desired, the reaction conditions must be adjusted again because all molecules take part in the reaction and may influence the required synthesis conditions and the properties of the particles. Moreover, the shape of the calcium phosphate based nanoparticles plays an important role regarding cell viability. The toxic effect of calcium phosphate nanoparticles was investigated for non-porous plate-, rod-, or needle-shaped, as well as spherical morphologies on two human cell lines (Zhao et al., 2013-a). Cell death was more pronounced with plate- and needle-shaped than with spherical- or rod-shaped structures at concentrations above 100 μg mL−1. In another publication cell death was observed with non-porous, rod-like particles on gastric cancer cells, cervical adenocarcinoma epithelial cells and hepatoma cells, whereas no cell death was observed for normal human hepatocyte cells at concentrations higher than 125 μg mL−1 (Tang et al., 2014). Below these high concentrations there has been no sign for cell death in either of these publications. The influence of the particle synthesis with respect to their toxicity was investigated in another publication on a macrophage cell line, and toxicity was highest for an autoclave synthesis approach with gel-like rod-shaped nanoapatite at concentrations higher than 125 μg mL−1 (Motskin et al., 2009). In contrast, nanocrystalline apatite is said to be biocompatible to a final concentration of 100 μg mL−1 (Delgado-Lopez et al., 2012). Therefore, the definition of biocompatibility with respect to calcium phosphate based nanoparticles still appears to be controversial.
In some instances, citric acid has been used as a synthetic aid for the preparation of crystalline calcium phosphate particles and nanoparticles. In the corresponding prior art, none of these particles and nanoparticles combine amorphous structure and internal mesoporosity. In contrast, they form agglomerates or aggregates having textural pores between the constituent domains of the agglomerates or aggregates. This textural porosity differs significantly from mesoporosity resulting from holes inside individual particles.
For example, Mitsionis et al. (2010) disclose the effect of citric acid on the synthesis of high temperature-sintered calcium phosphate ceramics, consisting of crystalline calcium phosphate with textural porosity.
For example, Chinese patent application no. CN 104 355 297 A describes crystalline calcium phosphate, namely hydroxyapatite, particles or nanoparticles that are made of dried bulk material consisting of intergrown nanoscale crystallites. CN 104 355 297 A describes a method for synthesizing a microemulsion of hydroxyapatite, wherein said method utilizes cetyltrimethylammonium bromide (CTAB) or citric acid as tension-active agent, which is not used as structure directing template. The resulting bulk material consists of inorganic materials without the included organic materials of a hybrid compound. Textural porosity generated by agglomeration of crystalline particles with aperture diameters of 19.56-40.13 nm are described. The surface structure of the crystalline bulk material thus contains “holes” at which compounds or drugs can be absorbed. Therefore, the generated pores are not within the single colloidal stable nanoparticles but result from the agglomeration of intergrown nanoscale domains at the external surface.
For example, Chinese patent application no. CN 101 428 779 A describes hollow nanostructured crystalline hydroxyapatite and a preparation method thereof. Citric acid and EDTA are used as sequestrants to avoid precipitation of calcium phosphate at pH 5.2.
Furthermore, Jacobs et al. (2013) disclose non-porous sodium citrate stabilized calcium phosphate nanoparticles for the sustained delivery of the chemotherapeutic agent cisplatin. In these nanoparticles citrate is loosely attached and/or coordinated to the outer surface of the nanoparticles and protects and/or stabilizes the calcium phosphate, e.g. from agglomeration or degradation. The chemotherapeutic agent cisplatin is only absorbed on the outer surface of the nanoparticles without a functional triggered release mechanism.
There is a need in the art for improved means and methods for targeted delivery of compounds, in particular for biocompatible drug delivery systems, such as for targeted and controlled release particularly in cancer treatment.